Alkaline phosphatase

ABSTRACT

A novel alkaline phosphatase and a method for the production of the alkaline phosphatase are described. A method for detecting a ligand in a sample and a method for analyzing the sequence of nucleic acid in a sample using the alkaline phosphatase are also described.

The present invention relates to novel alkaline posphatase whichexhibits high purity and good stability, to a method of manufacturingthe same and to a method of detecting a ligand in a sample using saidenzyme as a marker as well as a reagent used therefor.

Many methods have been known for detecting the biological substancesand, an analytical method utilizing a biochemical affinity has been usedto measure specifically a small amount of component in varioussubstances which are present in a mixed state. For example, with regardto a component such as glucose, uric acid and the like existing in aconcentration of 10⁻² mole/liter or more in body fluid, a detectingmethod utilizing an enzymatic reaction in which said component is asubstrate is frequently used, while, with regard to a component havinghigher molecular weight which cannot be used as a substrate or thathaving lower concentration, it is common to utilize ligand-receptorreaction such as antigen-antibody, hormone-receptor and nucleicacid-nucleic acid, which exhibit higher affinity for each component.

In such a detecting method, it is in many cases necessary that one ofthe affinity components, ligand or receptor is labeled for thedetection. As one of such detecting methods, a method with a radioactivesubstance or radioisotope (RI) is excellent in terms of detectionsensitivity and has been used already. However, it requires equipmentsand measuring devices by which specific radioisotopes are handled.Accordingly, a method with an enzyme as a marker has been used in recentyears. In the method, one of the affinity components is labeled with anenzyme, and the enzymatic activity of the marker which is bonded as aresult of ligand-receptor reaction or is not bonded is measured wherebyanother affinity component is quantitatively determined.

A significant improvement in the detection sensitivity has beenattempted for changing the substrate used for detecting the labeledenzyme from a substrate for calorimetric method to that for fluorescentmethod or, furthermore, that for chemiluminescence method.

Examples of the conditions or requirements for labeled enzyme are that,in general, purity is high, stability is high, turnover is high,functional group which is apt to be labeled is contained, Km value tothe substrate is low, background is low and the substrate which issuitable for the detection is available. Examples of the applicableenzyme are alkaline phosphatase, β-galactosidase, glucose oxidase,glucose-6-phosphate dehydrogenase, peroxidase, β-lactamase, glucoamylaseand lysozyme and a big advantage of alkaline phosphatase among the aboveenzymes is that its background is lower than those of other enzymes andthat the substrates suitable for detection are available.

With regard to the substrates for alkaline phosphatase which aresuitable for detecting antigen, antibody or nucleic acid, p-nitrophenylphosphate, 5-bromo-4-chloro-3-indolyl phosphate, 4-methylumbelliferonephosphate and chemiluminescent dioxetanes (PPD, AMPPD, etc.) have beenused already. When visible light, fluorescence or luminescence resultedby the reaction of those substrates with alkaline phosphatase ismeasured, the amount of the biological substance can be determined.

With regard to the alkaline phosphatase which exhibits theabove-mentioned properties to the highest extent, that which is derivedfrom calf intestine may be exemplified and has been used most commonly.

The alkaline phosphatase derived from calf intestine has a specificactivity of not less than 3,000 U/mg and has a sugar chain and,therefore, it can be labeled by a periodic acid method whereby it issaid to be better than the enzymes derived from others. On the otherhand, however, said alkaline phosphatase has been known to exhibitlittle stability and, due to the sugar chain therein, a background isgenerated (Besman, M., Coleman, J. E., J. Biol. Chem. 260, 1190(1985);Japanese Laid-Open Patent Publication Sho-60/180584).

In addition, alkaline phosphatase derived from Escherichia coli shows agood stability and its sample having a high purity is easily availablebut, its specific activity is as low as 60 U/mg and is not suitable foruse as an enzyme for labeling whereby it has been used only as an enzymefor dephosphorylation in molecular biology (Reid, R. W., Wilson, I. B.in "The Enzymes", 3rd Edition, 373 (1971)).

For improving those enzymes, there has been an attempt in which theamino acids in alkaline phosphatase derived from E. coli are substitutedby means of a site-specific mutation (Japanese Laid-Open PatentPublication Hei-4/349881). However, the mutant alkaline phosphataseobtained there shows an only increase in 3.9 times as much in thespecific activity and is not comparable with that derived from calfintestine.

There have been other attempts for obtaining alkaline phosphatase havinga high specific activity from nature and the papers on the enzymederived from alkalophilic Bacillus species (Nomoto, M., et al., Agric.Biol. Chem., 52(7), 1643 (1988)) and that derived from Bacilluslicheniformis (Hulett, F. M., J. Gen. Microbiol., 132, 2387 (1986)) havebeen reported.

However, specific activity of the former is 1,650 U/mg and can be hardlysaid that it is as good as that derived from calf intestine. In thelatter, its specific activity is 2115.9 U/mg but the measurement of theenzymatic activity is conducted at 55° C. and there is a paper reportingthat the activity at the practical temperature of 37° C. is expected tobe not more than 70% thereof (Hulett, F. M., et al., Biochemistry,10(8), 1364 (1971)).

There has been a demand for obtaining an alkaline phosphatase frommicroorganisms which exhibits a higher purity, a higher stability and aspecific activity which is nearly as same as that of the enzyme derivedfrom calf intestine.

The present inventors have conducted an extensive study for solving theabove-mentioned problems and have found an alkaline phosphatase havingan excellent stability against heat and a high specific activity frommicroorganisms beloging the genus Bacillus whereupon the presentinvention has been achieved.

Thus, the present invention relates to an alkaline phosphatase havingthe following physical and chemical properties.

1. It catalyzes the following reaction:

Orthophosphoric acid monoester+H₂ O→Alcohol+Orthophosphoric acid

2. Activators and stabilizers: Mg⁺⁺ and Co⁺⁺

3. Thermal stability: Stable at least for 30 minutes when treated at pH7.5 and at 60° C.

4. Specific activity: at least 2,300 U/mg.

5. It has no sugar chain.

6. Molecular weight: 140,000-150,000 (gel filtration) 65,000-67,000(SDS-PAGE)

The term of "stable" in thermal stability is defined that residualactivity is not less than 80%.

One specific example of the present invention is an alkaline phosphatasehaving the following physical and chemical properties:

1. It catalyzes the following reaction:

Orthophosphoric acid monoester+H₂ O→Alcohol+Orthophosphoric acid.

2. Activators and stabilizers: Mg⁺⁺ and Co⁺⁺.

3. Thermal stability: not higher than 60° C. (at pH 7.5 for 30 minutes).

4. Optimum temperature: not lower than 60° C.

5. Stable pH: pH 6-9 (at 25° C. for 16 hours).

6. Optimum pH: pH 9-10.

7. Specific activity: at least 2,300 U/mg.

8. It has no sugar chain.

9. Km value: 0.34 mM (to p-nitrophenylphosphoric acid).

10. Molecular weight: 140,000-150,000 (gel filtration) 65,000-67,000(SDS-PAGE)

11. Substrate specificity: It acts on p-nitrophenyl phosphate,4-methylumbelliferone phosphate, NADP, DL-α-glycerophosphate,β-glycerophosphate, phenylphosphate, phosphoethanolamine andglucose-6-phosphate.

Another specific example of the present invention is an alkalinephosphatase having the following physical and chemical properties.

1. It catalyzes the following reaction:

Orthophosphoric acid monoester+H₂ O→Alcohol+Orthophosphoric acid.

2. Activators and stabilizers: Mg⁺⁺ and Co⁺⁺.

3. Thermal stability: not higher than 60° C. (at pH 7.5 for 30 minutes).

4. Optimum temperature: not lower than 60° C.

5. Stable pH: pH 6-9 (at 25° C for 16 hours).

6. Optimum pH: pH 9-10.

7. Specific activity: at least 2,300 U/mg.

8. It has no sugar chain.

9. Km value: 0.26 mM (to p-nitrophenyl phosphoric acid).

10. Molecular weight: 140,000-150,000 (gel filtration) 65,000-67,000(SDS-PAGE)

11. Substrate specificity: It acts on p-nitrophenyl phosphate,4-methylumbelliferone phosphate, NADP, DL-α-glycerophosphate,β-glycerophosphate, phenylphosphate, phosphoethanolamine andglucose-6-phosphate.

Still another specific example of the present invention is an alkalinephosphatase having the following physical and chemical properties:

1. It catalyzes the following reaction:

Orthophosphoric acid monoester+H₂ O→Alcohol+Orthophosphoric acid.

2. Activators and stabilizers: Mg⁺⁺ and Co⁺⁺.

3. Thermal stability: not higher than 70° C. (at pH 7.5 for 30 minutes).

4. Optimum temperature: not lower than 60° C.

5. Stable pH: pH 6-11 (at 25° C. for 16 hours).

6. Optimum pH: pH 9.5-10.

7. Specific activity: at least 2,300 U/mg.

8. It has no sugar chain.

9. Km value: 0.28 mM (to p-nitrophenyl phosphoric acid).

10. Molecular weight: 140,000-150,000 (gel filtration) 65,000-67,000(SDS-PAGE)

11. Substrate specificity: It acts on p-nitrophenyl phosphate,4-methylumbelliferone phosphate, NADP, DL-α-glycerophosphate,β-glycerophosphate, phenylphosphate, phosphoethanolamine,glucose-1-phosphate and glucose-6-phosphate.

The present invention also relates to a method of manufacturing analkaline phosphatase, characterized in that, a strain which belongs tothe genus Bacillus and has an ability of producing the alkalinephosphatase having the above-mentioned physical and chemical propertiesis cultured in a medium and the alkaline phosphotase is collected fromthe cultured product.

The present invention also relates to a method of detecting a ligand ina sample, characterized in that, an alkaline phosphatase having thefollowing physical and chemical properties as a marker.

1. It catalyzes the following reaction:

Orthophosphoric acid monoester+H₂ O→Alcohol+Orthophosphoric acid

2. Activators and stabilizers: Mg⁺⁺ and Co⁺⁺.

3. Thermal stability: It is stable at least for 30 minutes when treatedat pH 7.5 and 60° C.

4. Specific activity: at least 2,300 U/mg.

5. It has not sugar chain.

6. Molecular weight: 140,000-150,000 (gel filtration) 65,000-67,000(SDS-PAGE)

The present invention also relates to a reagent for detecting a ligandin a biological sample containing (i) a substance which specificallyreacts with a ligand labeled with an alkaline phosphatase having thefollowing physical and chemical properties or (ii) a ligand which islabeled with an alkaline phosphatase having the following physical andchemical properties and a substance which specifically reacts with aligand and (iii) a reagent for measuring an alkaline phosphatase.

1. It catalyzes the following reaction:

Orthophosphoric acid monoester+H₂ O→Alcohol+Orthophosphoric acid

2. Activators and stabilizers: Mg⁺⁺ and Co⁺⁺.

3. Thermal stability: It is stable at least for 30 minutes when treatedat pH 7.5 and 60° C.

4. Specific activity: at least 2,300 U/mg.

5. It has not sugar chain.

6. Molecular weight: 140,000-150,000 (gel filtration) 65,000-67,000(SDS-PAGE).

The present invention further relates to a reagent for detecting aligand in a biological sample containing (i) a substance having aspecific affinity for a ligand to which avidin or biotin is bonded, (ii)an alkaline phosphatase having the following physical and chemicalproperties to which biotin or avidin is bonded and (iii) a substancewhich measures an alkaline phosphatase.

1. It catalyzes the following reaction:

Orthophosphoric acid monoester+H₂ O→Alcohol+Orthophosphoric acid

2. Activators and stabilizers: Mg⁺⁺ and Co⁺⁺.

3. Thermal stability: It is stable at least for 30 minutes when treatedat pH 7.5 and 60° C.

4. Specific activity: at least 2,300 U/mg.

5. It has no sugar chain.

6. Molecular weight: 140,000-150,000 (gel filtration) 65,000-67,000(SDS-PAGE).

The present invention also relates to a method of quantitativedetermination of a ligand in a biological sample, characterized in that,an alkaline phosphatase having the following physical and chemicalproperties is used as a marker.

1. It catalyzes the following reaction:

Orthophosphoric acid monoester+H₂ O→Alcohol+Orthophosphoric acid

2. Activators and stabilizers: Mg⁺⁺ and Co⁺⁺.

3. Thermal stability: It is stable at least for 30 minutes when treatedat pH 7.5 and 60° C.

4. Specific activity: at least 2,300 U/mg.

5. It has no sugar chain.

6. Molecular weight: 140,000-150,000 (gel filtration) 65,000-67,000(SDS-PAGE).

The present invention also relates to a method of analysing the sequenceof nucleic acid in a sample, characterized in that, an alkalinephosphatase having the following physical and chemical properties as amarker.

1. It catalyzes the following reaction:

Orthophosphoric acid monoester+H₂ O→Alcohol+Orthophosphoric acid

2. Activators and stabilizers: Mg⁺⁺ and Co⁺⁺.

3. Thermal stability: It is stable at least for 30 minutes when treatedat pH 7.5 and 60° C.

4. Specific activity: at least 2,300 U/mg.

5. It has no sugar chain.

6. Molecular weight: 140,000-150,000 (gel filtration) 65,000-67,000(SDS-PAGE).

With regard to a source of the enzyme of the present invention, anysource such as animals, plants and microorganisms may be used so far asit is capable of producing the alkaline phosphatase having theabove-mentioned physical and chemical properties. Preferred ones are thebacteria of the genus Bacillus capable of producing the alkalinephosphatase having the above-mentioned properties and suitable examplesare Bacillus badius TE3592 (FERM BP-5329), Bacillus badius TE3593 (FERMBP-5330) and Bacillus badius TE3597 (FERM BP-5120). Incidentally,Bacillus badius TE3592 and Bacillus badius TE3593 are the strainsisolated from the soil collected at Yogo-cho, Ika-ku, Shiga Prefecturewhile Bacillus badius TE3597 is the strain isolated from the soilcollected at Takefu-shi, Fukui Prefecture and their mycologicalproperties are as follows.

(A) Bacillus badius TE3592

(a) Morphology

(1) Shape: short bacillus

(2) Size of cells: 0.6×1.8-3.0 μm

(3) Polymorphism of cells: none

(4) Motility: none

(5) Spores: Endospores were elliptic and observed at the center orterminals of the cells. Swelling of the endospores was not observed.

(b) State of growth on a medium.

(1) Bouillon agar plate medium: Light greyish yellow colonies wereformed when cultured at 30° C. for 24 hours. Circumferences of thecolonies were erose and convex. Surfaces were smooth having a luster andwere translucent.

(2) Bouillon liquid culture: Growth was normal and homogeneously turbid.Neither semimentation nor fairy ring was formed.

(3) Bouillon gelatin stab culture: Growth was normal and only upperparts grew in a filiform. Liquefaction of gelatin was weak.

(4) Litmus milk: No change in color. Milk was not solidified.

(5) MacConkey's agar medium: no growth.

(6) Phenylethyl alcohol agar medium: grew but poor.

(c) Physiological Properties.

(1) Gram stain: negative (-) or indefinite

(2) Reduction of nitrate: -

(3) Denitrification: -

(4) MR test: -

(5) VP test: -

(6) Production of indole: -

(7) Production of hydrogen sulfide: -

(8) Decomposition of starch: -

(9) Decomposition of casein: +

(10) Decomposition of gelatin: +

(11) Decomposition of tyrosine: +

(12) Decomposition of Tween 80: -

(13) Utilization of citric acid:

Koser's medium: -

Christensen's medium: -

(14) Utilization of inorganic nitrogen sources (carbon source wasdl-malic acid):

Sodium nitrate: -

Ammonium sulfate: +

Sodium glutamate: +

(15) Formation of dyes: -

(16) Urease: -

(17) Oxidase: +

(18) Catalase: +

(19) β-Glactosidase: -

(20) Arginine dihydrase: -

(21) Lysine carboxylase: -

(22) Ornithine carboxylase: -

(23) Tryptophane deaminase: -

(24) β-Glucosidase: -

(25) Exocellular DNase: -

(26) Ranges for growth:

    ______________________________________                                               Growth temperature:                                                           10° C.                                                                            -                                                                  20° C.                                                                            +                                                                  25° C.                                                                            +                                                                  30° C.                                                                            +                                                                  37° C.                                                                            +                                                                  40° C.                                                                            +                                                                  50° C.                                                                            -                                                                  Growth pH                                                                     pH 4       -                                                                  pH 7       +                                                                  pH 9       +                                                                  NaCl concentration                                                            2%         +                                                                  5%         +                                                           ______________________________________                                    

(27) Behavior to oxygen: aerophilic

(28) O-F Test (Hugh Leifson method): -(sugar was not decomposed)

(29) Production of acid and gas from sugars:

    ______________________________________                                                        Acid Gas                                                      ______________________________________                                        L-Arabinose       -      -                                                    D-Xylose          -      -                                                    D-Glucose         -      -                                                    D-Mannose         -      -                                                    D-Fructose        -      -                                                    D-Galactose       -      -                                                    Maltose           -      -                                                    Sucrose           -      -                                                    Lactose           -      -                                                    Trehalose         -      -                                                    D-Sorbitol        -      -                                                    D-Mannitol        -      -                                                    Inositol          -      -                                                    Glycerol          -      -                                                    Starch            -      -                                                    L-Rhamnose        -      -                                                    D-Melibiose       -      -                                                    D-Amygdalin       -      -                                                    ______________________________________                                    

(30) Utilization of organic compounds.

    ______________________________________                                        D-Glucose          -                                                          L-Arabinose        -                                                          D-Mannose          -                                                          D-Mannitol         -                                                          N-Acetyl-D-glucosamine                                                                           -                                                          Maltose            -                                                          Potassium gluconate                                                                              +                                                          n-Capric acid      -                                                          Adipic acid        -                                                          dl-Malic acid      +                                                          Phenyl acetate     -                                                          ______________________________________                                    

(B) Bacillus badius TE3593.

(a) Morphology

(1) Shape: short bacillus

(2) Size of cells: 0.4×1.3-2.8 μm

(3) Polymorphism of cells: none

(4) Motility: none

(5) Spores: Endospores were elliptic and observed at the center orterminals of the cells. Swelling of the endospores was not observed.

(b) State of growth on a medium.

(1) Bouillon agar plate medium: Light greyish yellow colonies wereformed when cultured at 30° C. for 24 hours. Circumferences of thecolonies were erose and convex. Surfaces were smooth having a luster andwere translucent.

(2) Bouillon liquid culture: Growth was normal and homogeneously turbid.Neither semimentation nor fairy ring was formed.

(3) Bouillon gelatin stab culture: Growth was normal and only upperparts grew in a filiform. Liquefaction of gelatin was weak.

(4) Litmus milk: No change in color. Milk was not solidified.

(5) MacConkey's agar medium: no growth.

(6) Phenylethyl alcohol agar medium: grew but poor.

(c) Physiological Properties.

(1) Gram stain: negative (-) or indefinite

(2) Reduction of nitrate: -

(3) Denitrification: -

(4) MR test: -

(5) VP test: -

(6) Production of indole: -

(7) Production of hydrogen sulfide: -

(8) Decomposition of starch: -

(9) Decomposition of casein: +

(10) Decomposition of gelatin: +

(11) Decomposition of tyrosine: +

(12) Decomposition of Tween 80: -

(13) Utilization of citric acid:

Koser's medium: -

Christensen's medium: -

(14) Utilization of inorganic nitrogen sources (carbon source wasdl-malic acid):

Sodium nitrate: -

Ammonium sulfate: +

Sodium glutamate: +

(15) Production of dyes: -

(16) Urease: -

(17) Oxidase: +

(18) Catalase: +

(19) β-Glactosidase: -

(20) Arginine dihydrase: -

(21) Lysine carboxylase: -

(22) Ornithine carboxylase: -

(23) Tryptophane deaminase: -

(24) β-Glucosidase: -

(25) Exocellular DNase: -

(26) Ranges for growth:

    ______________________________________                                               Growth temperature:                                                           10° C.                                                                            -                                                                  20° C.                                                                            +                                                                  25° C.                                                                            +                                                                  30° C.                                                                            +                                                                  37° C.                                                                            +                                                                  40° C.                                                                            +                                                                  50° C.                                                                            -                                                                  Growth pH                                                                     pH 4       -                                                                  pH 7       +                                                                  pH 9       +                                                                  NaCl concentration                                                            2%         +                                                                  5%         +                                                           ______________________________________                                    

(27) Behavior to oxygen: aerophilic

(28) O-F Test (Hugh Leifson method): -(sugar was not decomposed)

(29) Production of acid and gas from sugars:

    ______________________________________                                                        Acid Gas                                                      ______________________________________                                        L-Arabinose       -      -                                                    D-Xylose          -      -                                                    D-Glucose         -      -                                                    D-Mannose         -      -                                                    D-Fructose        -      -                                                    D-Galactose       -      -                                                    Maltose           -      -                                                    Sucrose           -      -                                                    Lactose           -      -                                                    Trehalose         -      -                                                    D-Sorbitol        -      -                                                    D-Mannitol        -      -                                                    Inositol          -      -                                                    Glycerol          -      -                                                    Starch            -      -                                                    L-Rhamnose        -      -                                                    D-Melibiose       -      -                                                    D-Amygdalin       -      -                                                    ______________________________________                                    

(30) Utilization of organic compounds.

    ______________________________________                                        D-Glucose          -                                                          L-Arabinose        -                                                          D-Mannose          -                                                          D-Mannitol         -                                                          N-Acetyl-D-glucosamine                                                                           -                                                          Maltose            -                                                          Potassium gluconate                                                                              +                                                          n-Capric acid      -                                                          Adipic acid        -                                                          dl-Malic acid      +                                                          Citric acid        -                                                          Phenyl acetate     -                                                          ______________________________________                                    

(C) Bacillus badius TE3597.

(a) Morphology

(1) Shape: short bacillus

(2) Size of cells: 1.0×3.3-4.0 μm

(3) Polymorphism of cells: none

(4) Motility: none

(5) Spores: Endospores were elliptic and observed at the center orterminals of the cells. Swelling of the endospores was not observed.

(b) State of growth on a medium.

(1) Bouillon agar plate medium: Light greyish yellow colonies wereformed when cultured at 30° C. for 24 hours. Circumferences of thecolonies were erose and convex. Surfaces were smooth having a luster andwere translucent.

(2) Bouillon liquid culture: Growth was normal and homogeneously turbid.Neither semimentation nor fairy ring was formed.

(3) Bouillon gelatin stab culture: Growth was normal and only upperparts grew in a filiform. Liquefaction of gelatin was weak.

(4) Litmus milk: No change in color. Milk was not solidified.

(5) MacConkey's agar medium: no growth.

(6) Phenylethyl alcohol agar medium: grew but poor.

(c) Physiological Properties.

(1) Gram stain: positive (+)

(2) Reduction of nitrate: negative (-)

(3) Denitrification: -

(4) MR test: -

(5) VP test: -

(6) Production of indole: -

(7) Production of hydrogen sulfide: -

(8) Decomposition of starch: -

(9) Decomposition of casein: +

(10) Decomposition of gelatin: +

(11) Decomposition of tyrosine: +

(12) Decomposition of Tween 80: -

(13) Utilization of citric acid:

Koser's medium: -

Christensen's medium: -

(14) Utilization of inorganic nitrogen sources (carbon source wasdl-malic acid):

Sodium nitrate: -

Ammonium sulfate: +

Sodium glutamate: +

(15) Production of dyes: -

(16) Urease: -

(17) Oxidase: +

(18) Catalase: +

(19) β-Glactosidase: -

(20) Arginine dihydrase: -

(21) Lysine carboxylase: -

(22) Ornithine carboxylase: -

(23) Tryptophane deaminase: -

(24) β-Glucosidase: -

(25) Exocellular DNase: -

(26) Ranges for growth:

    ______________________________________                                               Growth temperature:                                                           10° C.                                                                            -                                                                  20° C.                                                                            +                                                                  25° C.                                                                            +                                                                  30° C.                                                                            +                                                                  37° C.                                                                            +                                                                  40° C.                                                                            +                                                                  50° C.                                                                            -                                                                  Growth pH                                                                     pH 4       -                                                                  pH 7       +                                                                  pH 9       +                                                                  NaCl concentration                                                            2%         +                                                                  5%         +                                                           ______________________________________                                    

(27) Behavior to oxygen: aerophilic

(28) O-F Test (Hugh Leifson method): -(sugar was not decomposed)

(29) Production of acid and gas from sugars:

    ______________________________________                                                        Acid Gas                                                      ______________________________________                                        L-Arabinose       -      -                                                    D-Xylose          -      -                                                    D-Glucose         -      -                                                    D-Mannose         -      -                                                    D-Fructose        -      -                                                    D-Galactose       -      -                                                    Maltose           -      -                                                    Sucrose           -      -                                                    Lactose           -      -                                                    Trehalose         -      -                                                    D-Sorbitol        -      -                                                    D-Mannitol        -      -                                                    Inositol          -      -                                                    Glycerol          -      -                                                    Starch            -      -                                                    L-Rhamnose        -      -                                                    D-Melibiose       -      -                                                    D-Amygdalin       -                                                           ______________________________________                                    

(30) Utilization of organic compounds.

    ______________________________________                                        D-Glucose          -                                                          L-Arabinose        -                                                          D-Mannose          -                                                          D-Mannitol         -                                                          N-Acetyl-D-glucosamine                                                                           +                                                          Malatose           -                                                          Potassium gluconate                                                                              -                                                          n-Capric acid      -                                                          Adipic acid        -                                                          dl-Malic acid      +                                                          Citric acid        -                                                          Phenyl acetate     +                                                          ______________________________________                                    

Method of experiment for identifying the above-mentioned mycologicalproperties was carried out mostly in accordance with "Classification andIdentification of Microorganisms" (Revised Edition) edited by TakejiHasegawa (Gakkai Shuppan Center; 1985). As a standard for classificationand identification, "Bergey's Manual of Systematic Bacteriology" (1984)was referred to.

When the above-mentioned literature and mycological properties arereferred to, it is believed that they belong to the genus Bacillusbecause, though there is a difference in terms of production ofexocellular DNase, all of them are aerophilic bacilli which are unstableto Gram stain and are capable of producing the spores. When the factsthat endospores are elliptic and do not sweel, no acid is produed fromD-glucose and, though geletin is decomposed, starch is not decomposedare taken into consideration, it is belived that all of them belong toBacillus badius in the genus Bacillus and they were named as Bacillusbadius TE3592, Bacillus badius TE 3593 and Bacillus badius TE3597,respectively.

Remarks:

(1) Bacillus badius TE3597 has been deposited on Jun. 1, 1995 under theBudapest Treaty under the Deposit No. FERM BP-5120 while Bacillus badiusTE3592 and Bacillus badius TE3593 have been deposited on Dec. 2, 1994(domestic deposit) under Deposit Nos. FERM P-14683 and FERM P-14684,respectively, and have been converted into international deposit on Dec.7, 1995 under Deposit Nos. FERM BP-5329 and FERM BP-5330, respectively.In all cases, the International Depositary Authority is NationalInstitute of Science and Human-Technology, Agency of Industrial Scienceand Technology, of 1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken 305,Japan.

In the manufacture of the enzyme of the present invention, theabove-mentioned microorganims capable of producing alkaline phosphataseis cultured in a nutrient medium and the alkaline phosphatase iscollected from said cultured medium. With regard to a medium used forthe culture of the alkaline phosphatase-productive microorganisms, anyof synthetic and natural media may be used so far as it containssuitable amounts of carbon sources, nitrogen sources, inorganicsubstances and other nutrients which can be utilized by the strain used.Examples of the carbon sources applicable are glucose and glycerol.Examples of the nitrogen sources applicable are nitrogen-containingnatural substances such as peptones, meat extract and yeast extract andinorganic nitrogen-containing compounds such as ammonium chloride andammonium citrate. Examples of the inorganic substances applicable arepotassium phosphate, sodium phosphate and magnesuim sulfate.Incidentally, it is desirable that the concentration of phosphoric acidis made low for inducing the production of alkaline phosphatase.

Culturing is usually conducted by a shake culture or by a culture withaeration and stirring. It is recommend to control the culturingtemperature to 20°-40° C. or, preferably, 25°-37° C. and the culturingpH to 5-11 or, preferably, 6-10. The conditions outside of the aboverange may be conducted as well provided that the used strain can grow.With regard to the culturing time, growth is resulted usually for 1-7days whereupon alkaline phosphatase is produced and accumualted bothinside and outside of the cells.

With regard to a method for purifying the enzyme of the presentinvention, that which has been commonly used may be used. For example,the medium after removal of the cells can be purified by means of asalting-out method using ammonium sulfate or sodium sulfate, a metalaggregating method using magnesium chloride or calcium chloride, anaggregating method using protamine or polyethyleneimine or an ionexchange chromatographic method using DEAE (diethylaminoethyl) celluloseor CM (carboxymethyl) Sepharose. Crude enzymatic liquid or purifiedenzymatic liquid obtained by those methods may also be pulverized, forexample, by a spray drying or a freeze drying. Further, it may be usedas an immobilized enzyme by immobizing with a suitable carrier.

Then, a method of measuring the activity of the alkaline phosphatase ofthe present invention will be given as hereunder.

First, the following reaction mixture liquid is prepared in a cuvetteand preliminarily warmed at 37° C. for about five minutes.

3.00 ml of 1M diethanolamine buffer (pH: 9.8) containing 0.1 mM of CoCl₂and 0.5 mM of MgCl₂ ; and

0.05 ml of 0.67M p-nitrophenyl phosphate buffer

Then 0.05 ml of an enzyme solution is added thereto followed by gentlemixing, changes in the absorbance at 405 nm are recorded for 3-4 minutesby a spectrophotometer controlled at 37° C. using water as a controland, from its initial linear part, changes in the absorption per minuteare determined. A blind test is conducted as follows. Thus, a dilutedenzyme solution (50 mM Tris hydrochloride buffer of pH 7.5 containing0.05 mM of CoCl₂ and 0.05 mM of MgCl₂) instead of the enzyme solution isadded to the reaction mixture solution, then the same operations asmentioned above are conducted and absorbance per minute is determined.Amount of the enzyme which produces one micromole of p-nitrophenol perminute under the above conditions is defined as one unit (U).

A specific method for detecting a ligand in the sample of the presentinvention is a method in which an affinity reaction of the ligand with asubstance having a specific affinity for the ligand is utilized and theactivity of the alkaline phosphatase bonded with the substance which isformed by said reaction is determined or is a method in which theactivity of the alkaline phosphatase which is not bonded therewith isdetermined.

Examples of the ligand in the sample in the present invention areantigen, antibody, hormone, hormone receptor or nucleic acid.

Examples of the affinity reaction of ligand with a substance having aspecific affinity for the ligand are antigen-antibody reaction,hormone-hormone receptor reaction and nucleic acid hybridation reaction.

With regard to the alkaline phosphatase which is used for the presentinvention, that of any source may be used so far as it is an alkalinephosphatase having the above-mentioned physical and chemical properties.A suitable example is an alkaline phosphatase of the genus Bacillus. Itsexamples are the alkaline phosphatases of Bacillus badius TE3592 andBacillus badius TE3593 and that of Bacillus badius TE3597.

Physical and chemical properties of the alkaline phosphatase of Bacillusbadius TE3492 are as follows.

1. It catalyzes the following reaction:

Orthophosphoric acid monoester+H₂ O→Alcohol+Orthophosphoric acid

2. Substrate specificity: It acts on p-nitrophenyl phosphate,4-methylumbelliferone phosphate, NADP, DL-α-glycerophosphate,β-glycerophosphate, phenylphosphate, phosphoethanolamine andglucose-6-phosphate.

3. Km value: 0.34 mM (to p-nitrophenyl phosphoric acid).

4. Optimum pH: pH 9-10.

5. Stable pH: pH 6-9 (at 25° C. for 16 hours).

6. Optimum temperature: not lower than 60° C.

7. Activators and stabilizers: Mg²⁺ and Co²⁺.

8. Specific activity: at least 2,300 U/mg.

9. It has no sugar chain.

10. Thermal stability: not higher than 60° C. (at pH 7.5 for 30minutes).

11. Molecular weight: 140,000-150,000 (gel filtration) 65,000-67,000(SDS-PAGE).

Physical and chemical properties of the alkaline phosphatase of Bacillusbadius TE3493 are as follows.

1. It catalyzes the following reaction:

Orthophosphoric acid monoester+H₂ O→Alcohol+Orthophosphoric acid

2. Substrate specificity: It acts on p-nitrophenyl phosphate,4-methylumbelliferone phosphate, NADP, DL-α-glycerophosphate,β-glycerophosphate, phenylphosphate, phosphoethanolamine andglucose-6-phosphate.

3. Km value: 0.26 mM (to p-nitrophenyl phosphoric acid).

4. Optimum pH: pH 9-10.

5. Stable pH: pH 6-9 (at 25° C. for 16 hours).

6. Optimum temperature: not lower than 60° C.

7. Activators and stabilizers: Mg²⁺ and Co²⁺.

8. Specific activity: at least 2,300 U/mg.

9. It has no sugar chain.

10. Thermal stability: not higher than 60° C. (at pH 7.5 for 30minutes).

11. Molecular weight: 140,000-150,000 (gel filtration) 65,000-67,000(SDS-PAGE).

Physical and chemical properties of the alkaline phosphatase of Bacillusbadius TE3497 are as follows.

1. It catalyzes the following reaction:

Orthophosphoric acid monoester+H₂ O→Alcohol+Orthophosphoric acid

2. Substrate specificity: It acts on p-nitrophenyl phosphate,4-methylumbelliferone phosphate, NADP, DL-α-glycerophosphate,β-glycerophosphate, phenylphosphate, phosphoethanolamine,glucose-1-phospohate and glucose-6-phosphate.

3. Km value: 0.28 mM (to p-nitrophenyl phosphoric acid).

4. Optimum pH: pH 9.5-10.

5. Stable pH: pH 6-11 (at 25° C. for 16 hours).

6. Optimum temperature: not lower than 60° C.

7. Activators and stabilizers: Mg²⁺ and Co²⁺.

8. Specific activity: at least 2,300 U/mg.

9. It has no sugar chain.

10. Thermal stability: not higher than 70° C. (at pH 7.5 for 30minutes).

11. Molecular weight: 140,000-150,000 (gel filtration) 65,000-67,000(SDS-PAGE).

Examples of the substance which labels the alkaline phosphatase in thepresent invention are antigens which are substances having a highmolceular weight such as protein and nucleic acid. Peptides in which anepitope site of antigen is designed which has been much used recentlymay be used as well. With regard to antibody, the commonly-used onessuch as polyclonal antibody obtained by immunization to goats, rabbitsand guinea pigs, monoclonal antibody obtained from hybridoma ofabdominal dropsy of mouse and antigen-bonded active fragment (Fab')prepared by treating those antibodies with protease may be used. It isalso possible to use protein having antigen-bonding activity such as Fvantibody and single stranded Fv antibody obtained by a gene recombinanttechnique.

In the present invention, it is preferred that the alkaline phosphataseis bonded with any of a ligand and a substance which has a specificaffinity for the ligand.

With regard to a method which is used for labeling the alkalinephosphatase with the above-mentioned antigen or antibody, glutaraldehydemethod, maleimide method, carbodiimide method, pyridine disulfidemethod, etc. may be used and the preferred ones are maleimide method orthe like in which the activity of antigen, antibody and enzyme is notlowered.

With regard to the enzyme for labeling which is to be introduced intoone molecule of antibody or antigen, it is usually preferred to use anenzyme marker to which one or more molecule(s), preferably two or moremolecules, is/are bonded.

One specific example of the present invention is that a ligand in thesample such as antigen or antibody is made to react with a substancebonded with an alkaline phosphatase and having a specific affinity forsaid ligand such as antibody or antigen, reaction products and unreactedsubstances are separated and the activity of the alkaline phosphatasebonded with the reaction products or the activity of the alkalinephosphatase of the unreacted substances is measured.

The immunoasssay using an alkaline phosphatase as a labeled substancecan be used for a competitive method or a heterogenous method of anoncompetitive method in the measurement of both antibody and antigen.In any of the methods, it is possible to label an alkaline phosphatasethereto not only in a method using primary antibody but also in a methodusing secondary antibody. Further, instead of the secondary antibody, Fcreceptor such as protein A and protein G can be used by labeling with analkaline phosphatase.

Another specific example of the present invention is a method ofdetecting a ligand in which a ligand or a substance having a specificaffinity for the ligand is bonded to a solid phase. With regard to thesolid phase, the conventionally known ones such as polystyrene beads maybe used.

Still another specific example is a method of detecting a ligand in asample in which avidin or biotin is bonded with a substance (e.g.antibody or antigen) having a specific affinity for a ligand whilealkaline phosphatase is bonded with biotin or avidin and, simultaneouslywith or after an affinity reaction of a ligand with a substance (e.g.antibody or antigen) having a specific affinity for the ligand, areaction of binding avidin with biotin is conducted and the alkalinephosphatase activity bonded by said reaction or the residual alkalinephosphatase activity is measured.

Avidin compounds are sugar proteins which can bind with biotin stronglyand are exampled by avidin, streptoavidin and the like. And biotincompounds are ones of vitamine B complexes and biotin is represented.

Binding constants of avidin and biotin are as high as in a level of 10¹⁵M⁻¹, avidin has four sites for binding with biotin and introduction ofbiotin has little loss in the activity of, for example, antibody.Accordingly, an avidin-biotin system has a big advantage.

In a solid phase sandwich immunoassay for example, antibody which isimmobilized with a solid phase is made to react with a sample, then madeto react with antibody which is bonded with biotin and, after that,biotin in the reaction product or that in the unreacted substance isdetected by avidin which is labeled with alkaline phosphatase. Avidinand streptoavidin which is a analogous substance thereof are proteinswith a molecular weight of about 50,000 and they can be bonded withalkaline phosphatase by glutaraldehyde method, maleimide method,carbodiimide method, pyridine disulfide method, etc.

On the other hand, when an antibody bonded with avidin instead of biotinis used, a detection is conducted with biotin which is labeled withalkaline phosphatase and avidin after the antigen-antibody reaction.Reagents which biotinizes the alkaline phosphatase are available inmarket and their examples are biotin-N-hydroxysuccinimide ester,biotin-N-hydroxysulfosuccinimide ester and biotinoyl-ε-aminocaproic acidN-hydroxysuccinimide ester.

A reagent for detecting a ligand in a sample in accordance with thepresent invention contains a substance which specifically bonds with aligand labeled by an alkaline phosphatase having the following physicaland chemical properties or a ligand labeled by an alkaline phosphatasehaving the following physical and chemical properties and a reagent foridentifying the alkaline phosphatase. Examples of the reagent fordetecting the alkaline phosphatase are coloring substrate, fluorescentsubstrate and luminous substrate.

Examples of the coloring substrate according to the present inventionare p-nitrophenyl phosphate, 1-naphtholphthalein monophosphate (JapaneseLaid-Open Patent Publication Hei-5-13958), 5-bromo-4-chloro-3-indolylphosphate and a combination thereof with nitro blue tetrazolium.

Examples of the fluorescent substrate are 4-methylumbelliferonephosphate, phenalenone-6-phosphate and analogous compounds thereof andbenzphenalene-6-phosphate and analogous compounds thereof (JapaneseLaid-Open Patent Publication Sho-62/190191).

Examples of the luminous substrate are 1,2-dioxetane compounds such asPPD and AMPPD as well as derivatives thereof and mixture thereof withenhancers such as surface-active agents, fluorescent substances andproteins.

Concentration of those substrates of the present invention is 0.01-200mmoles/l or, preferably, 0.05-50 mmoles/l. The enzymatic reaction of thepresent invention is usually carried out at pH 7-11 but, when an optimumpH is taken into consideration, it is desirable to conduct the enzymaticreaction at pH 8-11. Examples of the buffer applicable are Trishydrochloride buffer, phosphate buffer, diethanolamine hydrochloridebuffer, triethanolamine hydrochloride buffer, bicarbonate buffer,N-methyl-D-glucamine hydrochloride buffer, barbital buffer, glycinesodium hydroxide buffer, 2-amino-2-methylpropanol hydrochloride bufferand amino alcohol type buffer. Concentration of those buffers is 5-200mmoles/l or, preferably, 20-500 mmoles/l.

Though activity of the enzyme bonded with the reaction product or thatbonded with the unreacted substance can be determined by measuring theactivity of alkaline phosphatase by a rate method, it can be alsodetermined by the reaction of the above-mentioned substrate with thesubstance to which the enzyme is bonded followed by detecting saidproduct after stopping the reaction. Examples of the applicable agentfor stopping the reaction are alkaline solutions, enzyme inhibitors,chelating agents such as EDTA and inorganic phosphoric acids. When thereaction system is made strongly alkaline after stopping the reaction,sensitivity can be made higher in the case of the substrate such asp-nitrophenyl phosphate and dioxetane compounds (Japanese Laid-OpenPatent Publication Hei-2/273199).

It is desirable that metal salts are added to the buffer used for thepresent invention whereby inactivation of the alkaline phosphatase isprevented. Examples of the metal salts applicable are magnesium salt,cobalt salt, zinc salt, manganese salt and calcium salt and, preferably,magnesium salt and cobalt salt. Preferred concentration of the magnesiumsalt to be added is 0.05 mmole/l to 10 mmoles/l and examples of themagnesium salt applicable are magnesium acetate, magnesium chloride,magnesium citrate, magnesium sulfate and magnesium complexes such asmagnesium ethylenediamine tetraacetate. Preferred concentration of thecobalt salt to be added is 0.02-5 mmoles/l and examples of theapplicable cobalt salt are cobalt acetate, cobalt chloride, cobaltcitrate, cobalt sulfate and cobalt complexes such as cobaltethylenediamine tetraacetate. A joint use of magnesium salt and cobaltsalt is desirable but it is not essential.

With regard to the surface-active agent which is useful for conductingthe present invention, any agent which does not too much inhibit theactivity of alkaline phosphatase may be used. Generally, usefulsurface-active agents are nonionic surface-active agents thoughamphoteric and ionic ones may be used as well.

An organic solvent which is miscible with water is also possibly usedtogether in the present invention. Examples of the organic solvent aremethanol, ethanol, propanol, N,N-dimethylformamide, dimethyl sulfoxide,acetonitrile and hexamethylene phosphamide.

Other compounds may be also added to the reagent of the presentinvention to conduct the enzymatic reaction smoothly or to keep theactivity of the constituting components. Examples of such compounds arestabilizers and excipients. Further, addition of an inactivated typeenzyme of alkaline phosphatase is effective for reducing the backgroundand non-specific reactions.

The present invention can be applied not only to the above-mentionedsolid phase sandwich immunoassay but also to a measurement of hormoneand its receptor utilizing an affinity between hormone and receptor andto a measurement utilizing an affinity between nucleic acid and nucleicacid such as DNA-DNA reaction and DNA-RNA reaction.

A specific example of the present invention for detecting the nucleicacid is that a sample containing nucleic acid (DNA or RNA) is made toact on a material to which the captured oligonucleotide is immobilized,then the detected oligonucleotide which is labeled with an alkalinephosphatase is made to act whereupon a nucleic acid hybridization of thenucleic acid bonding the captured oligonucleotide with the detectedlabeled oligonucleotide is conducted and, after separating the unreacteddetected oligonucleotide, the activity of the alkaline phosphatase ofthe substance to which a nucleic acid hybridization is carried out ismeasured whereby the nucleic acid in the sample is detected.

Examples of the nucleic acid in a sample are single- and double-strandednucleic acid such as DNA and RNA. Examples of the sample are body fluidsuch as serum, urine and lymph as well as other materials such asvarious tissues.

In the preparation of the complex of the alkaline phosphatase with DNAor RNA according to the present invention, a method mentioned, forexample, in M. Renz and C. Kurz, Nucleic Acids Res., 12(8), 343 (1984)may be applied. For example, alkaline phosphatase is cross-linked withpolyethyleneimine and the resulting conjugate is cross-linked witholigonucleotide of DNA or RNA using glutaraldehyde to give a labelednucleic acid.

Reagents for a direct introduction of amino group or thiol group via aspacer arm to the 5'-terminal or any other chain in the synthesis ofoligonucleotides are available in market. It is possible to introduce analkaline phosphatase into oligonucleotide by bonding such a reagent withthe alkaline phosphatase by, for example, means of glutaraldehydemethod, maleimide method, carbodiimide method, succinimide ester methodand pyridine disulfide method.

An example of the sample is DNA and, when an elongating reaction iscarried out using DNA as a template, biotin is incorporated into DNAfragments utilizing a biotinized primer or a biotinized terminator.

After that, the fragments are developed by electrophoresis and then theyare made to react with an avidinized alkaline phosphatase or with avidinand then with a biotinized alkaline phosphatase to detect theabove-mentioned fragments.

Another specific example of the present invention is a method ofanalysing the sequence of the nucleic acid in the sample, characterizedin that, the alkaline phosphatase having the above-mentioned physicaland chemical properties is used as a marker. To determine the sequenceof nucleic acids, one can select one of conventional methods which aredideoxytermination, Maxam-Gilbert and so forth. The following proceduresare exampled in order to carry out the dideoxytermination method; First,biotin primers are hybridised with the nucleic acids to be determinedthat are either double-strands or single-strand denatured by alkalinesolution, adding a dideoxyribonucleotide (ex. ddATP), fourdeoxyribonucleotides (dNTPs) and nucleic acid synthesis enzymes (ex.Klenow, T7 DNA polymerase and the like) to proceed the primer-extensionreaction and terminate the reaction simultaneously. Then, another threeyribonucleotides (ddCTP, ddGTP and ddTTP) are used in the same reactionas that with the above described ddATP. After subjecting the resultingfour reactants to an electrophoresis, alkaline phosphatase with avidinor avidin and then alkaline phosphatase with biotin are reacted with thereactants to detect the extended fragments using chemiluminescencesubstrates and so forth and determine comparatively the sequence froneach lane electrophoresed.

The reagent for analysing the nucleic acid sequence using theabove-mentioned nucleic acid which is labeled with alkaline phosphataseincludes a reagent for measuring the alkaline phosphatase and DNA or RNAwhich is bonded with said enzyme.

The present invention is further applicable to an in situ hybridizationwherein a nucleic acid hybridization is conducted in cells instead ofconducting a nucleic acid hybridization after taking out the nucleicacid from the sample.

According to the present invention, it is possible to afford a novelalkaline phosphatase which exhibits high purity and good stability.

A reagent composition for a binding assay using the alkaline phosphataseof the present invention exhibits a high sensitivity and an excellentstorability for long time. In addition, it gives good results withlittle background in the detection of the aimed substances.

This invention will be further explained by means of the followingExamples which refer partly to the accompanying drawings wherein:

FIG. 1 is a graph showing the relation between the reaction pH andrelative activity of the enzyme produced by Bacillus badius TE3592,

FIG. 2 is a graph showing the pH stability of the enzyme produced byBacillus badius TE3592,

FIG. 3 is a graph showing the relation between the reaction temperatureand the relative activity of the enzyme produced by Bacillus badiusTE3592,

FIG. 4 is a graph showing the thermal stability of the enzyme producedby Bacillus badius TE3592,

FIG. 5 is a graph showing the relation between the reaction pH and therelative activity of the enzyme produced by Bacillus badius TE3593,

FIG. 6 is a graph showing the pH stability of the enzyme produced byBacillus badius TE3593,

FIG. 7 is a graph showing the relation between the reaction temperatureand the relative activity of the enzyme produced by Bacillus badiusTE3593,

FIG. 8 is a graph showing the thermal stability of the enzyme producedby Bacillus badius TE3593,

FIG. 9 is a graph showing the relation between the reaction pH and therelative activity of the enzyme produced by Bacillus badius TE3597,

FIG. 10 is a graph showing the pH stability of the enzyme produced byBacillus badius TE3597,

FIG. 11 is a graph showing the relation between the reaction temperatureand the relative activity of the enzyme produced by Bacillus badiusTE3597,

FIG. 12 is a graph showing the thermal stability of the enzyme producedby Bacillus badius TE3597,

FIG. 13 shows a comparison of calibration curves of human CRP using goatanti-human CRPIgG labeled with the enzyme of the present invention andthat labeled with CIAP,

FIG. 14 shows a comparison of calibration curves of human CRP using goatanti-human CRP Fab' labeled with the enzyme of the present invention andthat labeled with CIAP,

FIG. 15 shows a comparison of the stabilities upon storage ofstreptoavidin labeled with the enzyme of the present invention and thatlabeled with CIAP,

FIG. 16 shows a comparison of thermal stabilities of biotin labeled withthe enzyme of the present invention and that labeled with CIAP, and

FIG. 17 shows thermal stability of the probe labeled with the enzyme ofthe present invention and that labeled with CIAP.

EXAMPLE 1

A medium (100 ml) containing 3.0% of glycerol, 1.0% of polypeptone, 0.1%of yeast extract, 0.02% of magnesium sulfate, 0.002% of monopotassiumphosphate and 0.3% of sodium chloride was transferred to a 500 mlSakaguchi's flask and autoclaved at 121° C. for 15 minutes. One platinumloop of Bacillus badius TE3592 (FERM BP-5329) was inoculated as a seedand cultured at 30° C. for 20 hours to prepare a seed culture liquid.Then six liters of the same medium was transferred to a 10 liter jarfermentor, autoclaved at 121° C. for 15 minutes, allowed to cool and 100ml of the seed culture liquid was added thereto followed by culturing at300 rpm and 30° C. with 2 liters/minute of aeration for 20 hours. Theresulting culture liquid was centrifuged to give a supernatant liquid.This liquid was purified to an extent of 2,300 U/mg of specific activityby means of fractionation with sodium sulfate, DEAE-Sepharosechromatography, phenyl Sepharose chromatography and gel filtration withSephadex G-200.

The resulting alkaline phosphatase had the following characteristics.

1. It catalyzed the following reaction.

Orthophosphoric acid monoester+H₂ O→Alcohol+Orthophosphoric acid

2. Substrate specificitiy. (shown in Table 1)

                  TABLE 1                                                         ______________________________________                                        Compounds          Relative Activity                                          ______________________________________                                        p-Nitrophenyl phosphate                                                                          100                                                        4-Methylumbelliferyl phosphate                                                                   87.6                                                       NADP               15.4                                                       DL-α-Glycerophosphate                                                                      37.9                                                       β-Glycerophosphate                                                                          49.7                                                       Phenyl phosphate   140                                                        Phosphoryl choline 2.96                                                       Phosphoethanolamine                                                                              44.4                                                       Glucose-1-phosphate                                                                              0                                                          Glucose-6-phosphate                                                                              14.8                                                       ______________________________________                                    

3. Km value.

The Km value to p-nitrophenol was 0.34 mM.

4. Optimum pH.

Its enzymatic activity in 0.97M diethanolamine buffer (pH: 8.0-11.0) wasmeasured. The result was as shown in FIG. 1 and the optimum pH was 9-10.

5. Stable pH.

It was stored in glycine hydrochloride buffer (pH: 2-3), acetate buffer(pH: 3-6), potassium phosphate buffer (pH: 6-8), Tris hydrochloridebuffer (pH: 8-9) and glycine-NaOH buffer (pH: 9-10) at 25° C. for 16hours and the residual activities were measured. The result was as shownin FIG. 2 and the stable pH was 6-9.

6. Optimum temperature.

Its enzymatic activities at various temperature were measured. Theresult was as shown in FIG. 3 and the optimum temperature was not lowerthan 60° C.

7. Thermal stability.

The enzyme of the present invention was warmed for 30 minutes in a 50 mMTris hydrochloride buffer (pH: 7.5) containing 1.0 mM of MgCl₂ and 0.1mM of CoCl₂ and the residual enzymatic activity was measured. The resultwas as given in FIG. 4 and the enzyme was found to be stable until 60°C.

8. Activators and stabilizers.

Mg⁺⁺ and Co⁺⁺ were found to be essential.

9. Molecular weight.

140,000-150,000 (gel filtration) 65,000-67,000 (SDS-PAGE)

10. Sugar content.

No sugar was detected.

EXAMPLE 2

A medium (100 ml) containing 3.0% of glycerol, 1.0% of polypeptone, 0.1%of yeast extract, 0.02% of magnesium sulfate, 0.002% of monopotassiumphosphate and 0.3% of sodium chloride was transferred to a 500 mlSakaguchi's flask and autoclaved at 121° C. for 15 minutes. One platinumloop of Bacillus badius TE3593 (FERM BP-5330) was inoculated as a seedand cultured at 30° C. for 20 hours to prepare a seed culture liquid.Then six liters of the same medium was transferred to a 10 liter jarfermentor, autoclaved at 121° C. for 15 minutes, allowed to cool and 100ml of the seed culture liquid was added thereto followed by culturing at300 rpm and 30° C. with 2 liters/minute of aeration for 20 hours. Theresulting culture liquid was centrifuged to give a supernatant liquid.This liquid was purified to an extent of 2,790 U/mg of specific activityby means of fractionation with sodium sulfate, DEAE-Sepharosechromatography, phenyl Sepharose chromatography and gel filtration withSephadex G-200.

The resulting alkaline phosphatase had the following characteristics.

1. It catalyzed the following reaction.

Orthophosphoric acid monoester+H₂ O→Alcohol+Orthophosphoric acid

2. Substrate specificitiy. (shown in Table 2)

                  TABLE 2                                                         ______________________________________                                        Compounds          Relative Activity                                          ______________________________________                                        p-Nitrophenyl phosphate                                                                          100                                                        4-Methylumbelliferyl phosphate                                                                   108                                                        NADP               90.5                                                       DL-α-Glycerophosphate                                                                      71.4                                                       β-Glycerophosphate                                                                          78.1                                                       Phenyl phosphate   180                                                        Phosphoryl choline 3.81                                                       Phosphoethanolamine                                                                              73.8                                                       Glucose-1-phosphate                                                                              1.44                                                       Glucose-6-phosphate                                                                              44.3                                                       ______________________________________                                    

3. Km value.

The Km value to p-nitrophenol was 0.26 mM.

4. Optimum pH.

Its enzymatic activity in 0.97M diethanolamine buffer (pH: 8.0-11.0) wasmeasured. The result was as shown in FIG. 5 and the optimum pH was 9-10.

5. Stable pH.

It was stored in glycine hydrochloride buffer (pH: 2-3), acetate buffer(pH: 3-6), potassium phosphate buffer (pH: 6-8), Tris hydrochloridebuffer (pH: 8-9) and glycine-NaOH buffer (pH: 9-10) at 25° C. for 16hours and the remaining activities were measured. The result was asshown in FIG. 6 and the stable pH was 6-9.

6. Optimum temperature.

Its enzymatic activities at various temperature were measured. Theresult was as shown in FIG. 7 and the optimum temperature was not lowerthan 60° C.

7. Thermal stability.

The enzyme of the present invention was warmed for 30 minutes in a 50 mMTris hydrochloride buffer (pH: 7.5) containing 1.0 mM of MgCl₂ and 0.1mM of CoCl₂ and the remained enzymatic activity was measured. The resultwas as given in FIG. 8 and the enzyme was found to be stable until 60°C.

8. Activators and stabilizers.

Mg⁺⁺ and Co⁺⁺ were found to be essential.

9. Molecular weight.

140,000-150,000 (gel filtration) 65,000-67,000 (SDS-PAGE)

10 Sugar content.

No sugar was detected.

EXAMPLE 3

A medium (100 ml) containing 3.0% of glycerol, 1.0% of polypeptone, 0.1%of yeast extract, 0.02% of magnesium sulfate, 0.002% of monopotassiumphosphate and 0.3% of sodium chloride was transferred to a 500 mlSakaguchi's flask and autoclaved at 121° C. for 15 minutes. One platinumloop of Bacillus badius TE3597 (FERM BP-5120) was inoculated as a seedand cultured at 30° C. for 20 hours to prepare a seed culture liquid.Then six liters of the same medium was transferred to a 10 liter jarfermentor, autoclaved at 121° C. for 15 minutes, allowed to cool and 100ml of the seed culture liquid was added thereto followed by culturing at300 rpm and 30° C. with 2 liters/minute of aeration for 20 hours. Theresulting culture liquid was centrifuged to give a supernatant liquid.This liquid was purified to an extent of 2,300 U/mg of specific activityby means of fractionation with sodium sulfate, DEAE-Sepharosechromatography, phenyl Sepharose chromatography and gel filtration withSephadex G-200.

The resulting alkaline phosphatase had the following characteristics.

1. It catalyzed the following reaction.

Orthophosphoric acid monoester+H₂ O.increment.Alcohol+Orthophosphoricacid

2. Substrate specificitiy. (shown in Table 3)

                  TABLE 3                                                         ______________________________________                                        Compounds          Relative Activity                                          ______________________________________                                        p-Nitrophenyl phosphate                                                                          100                                                        4-Methylumbelliferyl phosphate                                                                   98.0                                                       NADP               15.4                                                       DL-α-Glycerophosphate                                                                      67.0                                                       β-Glycerophosphate                                                                          67.5                                                       Phenyl phosphate   167                                                        Phosphoryl choline 6.50                                                       Phosphoethanolamine                                                                              67.0                                                       Glucose-1-phosphate                                                                              25.0                                                       Glucose-6-phosphate                                                                              34.5                                                       ______________________________________                                    

3. Km value.

The Km value to p-nitrophenol was 0.28 mM.

4. Optimum pH.

Its enzymatic activity in 0.97M diethanolamine buffer (pH: 8.0-11.0) wasmeasured. The result was as shown in FIG. 9 and the optimum pH was9.5-10.

5. Stable pH.

It was stored in glycine hydrochloride buffer (pH: 2-3), acetate buffer(pH: 3-6), potassium phosphate buffer (pH: 6-8), Tris hydrochloridebuffer (pH: 8-9) and glycine-NaOH buffer (pH: 9-10) at 25° C. for 16hours and the residual activities were measured. The result was as shownin FIG. 10 and the stable pH was 6-11.

6. Optimum temperature.

Its enzymatic activities at various temperature were measured. Theresult was as shown in FIG. 11 and the optimum temperature was not lowerthan 60° C.

7. Thermal stability.

The enzyme of the present invention was warmed for 30 minutes in a 50 mMTris hydrochloride buffer (pH: 7.5) containing 1.0 mM of MgCl₂ and 0.1mM of CoCl₂ and the remained enzymatic activity was measured. The resultwas as given in FIG. 12 and the enzyme was found to be stable until 70°C.

8. Activators and stabilizers.

Mg⁺⁺ and Co⁺⁺ were found to be essential.

9. Molecular weight.

140,000-150,000 (gel filtration) 65,000-67,000 (SDS-PAGE)

10. Sugar content.

No sugar was detected.

Comparative Example 1.

Properties of the enzymes of the present invention were compared withthose of known enzymes in the following table.

                  TABLE 4                                                         ______________________________________                                                                     Bacillus                                                                              Bacillus                                          Calf      Bacillus sp.                                                                            liqueniformis                                                                         Badius                                            Intestine OK-1      MC14    TE3592                                   ______________________________________                                        Activators                                                                             Mg.sup.++ Co.sup.++ Co.sup.++                                                                             Mg.sup.++,Co.sup.++                      Thermal  ≦45° C.                                                                   ≦60° C.                                                                   ≦60° C.                                                                 ≦60° C.                    stability                                                                     Optimum  35-45° C.                                                                        50° C.     ≧60° C.                    temperature                                                                   Stable pH                                                                              6-11      5-12              6-9                                      Optimum pH                                                                             10        11                9-10                                     Specific 3000-     1650 U/mg 2115.9 U/mg                                                                           2300 U/mg                                activity (temp)                                                                        5000 U/mg (37° C.)                                                                         (55° C.)                                                                       (37° C.)                          Sugar chain                                                                            present   none      none    none                                     Km value           0.037 mM          0.34 mM                                  (p-nitrophenyl                                                                phosphate)                                                                    Molecular                                                                              80,000    110,000   110,000 140,000-                                 weight                               150,000                                  (gel filtration)                                                              References                                                                             JP Laid   Agr. Biol.                                                                              J. Gen. (this                                             Open Pat  Chem. 52  Microbiol.                                                                            invention)                                        Pub Sho-  (7)1643   132, 2387                                                 60/180584 (1988)    (1986)                                           ______________________________________                                                       Bacillus Badius                                                                            Bacillus Badius                                                  TE3593       TE3597                                            ______________________________________                                        Activators     Mg.sup.++,Co.sup.++                                                                        Mg.sup.++,Co.sup.++                               Thermal stability                                                                            ≦60° C.                                                                      ≦70° C.                             Optimum temperature                                                                          ≧60° C.                                                                      ≧60° C.                             Stable pH      6-9          6-11                                              Optimum pH     9-10         9.5-10                                            Specific activity                                                                            2790 U/mg    2300 U/mg                                         (temp)         (37° C.)                                                                            (37° C.)                                   Sugar chain    none         none                                              Km value       0.26 mM      0.284 mM                                          (p-nitrophenyl phosphate)                                                     Molecular weight                                                                             140,000-     140,000-                                          (gel filtration)                                                                             150,000      150,000                                           References     (this invention)                                                                           (this invention)                                  ______________________________________                                    

EXAMPLE 4

(1) Preparation of goad anti-human CRPIgG labeled with an alkalinephosphatase.

A 25% glutaraldehyde solution (35 μl) was added to 2.5 ml of 50 mMphosphate buffer (pH: 7.2; containing 1 mM MgCl₂ and 0.1 mM CoCl₂)containing 5 mg of the alkaline phosphatase of Referential Example 1 andan incubation was conducted at 25° C. for 50 minutes. Then 0.5 ml of 50mM phosphate buffer (pH: 7.2) containing 2.5 mg of goat anti-humanCRPIgG fraction (manufactured by Nippon Biotest Laboratory) was addedthereto and an incubation was conducted at 25° C. for 75 minutes more.After that, 150 μl of 2M Tris/HCl (pH: 8.7) was added, the mixture wasstirred at 4° C. for 30 minutes, 150 μl of aqueous solution containing150 mg of NaBH₄ was added and an incubation was conducted at 4° C. for15 hours. The resulting mixture was purified by means of a highperformance liquid chromatography using Superdex™ 200 (manufactured byFarmacia) (using 50 mM Tris/HCl pH: 8.0! as an eluting liquid containing0.1M NaCl, 1 mM MgCl₂, 0.1 mM CoCl₂ and 0.1% of NaN₃) and the first peakwas obtained as an enzyme-labeled antibody.

(2) Calibration curve of human CRP.

Human CRP (0-1000 ng/ml) (1 ml) was added to one polystyrene bead coatedwith goat anti-human CRPIgG fractions (manufactured by Nippon BiotestLaboratory) and an incubation was conducted at 30° C. for one hour. Thenthe solid phase was washed with PBS thrice, 1 ml of an enzyme-labeledantibody diluted to an extent of 1 U/ml in terms of an alkalinephosphatase activity was added and an incubation was conducted at 30° C.for one hours. This was further washed with PBS thrice, a 1Mdiethanolamine buffer (pH: 9.8) containing 11 mM p-nitrophenyl phosphateand 5 mM MgCl₂ was added, a reaction was conducted at 37° C. for 30minutes, 2 ml of 0.5N NaOH was added to stop the reaction and theabsorption at 405 nm was measured to prepare a calibration curve (FIG.13).

Comparative Example 2.

A 25% glutaraldehyde solution (150 μl) was added to 2.5 ml of a 50 mMphosphate buffer (pH: 7.2) containing 5 mg of an alkaline phosphatasederived from calf intestine and the same operations as in Example 1 wereconducted to prepare an enzyme-labeled antibody.

The above-prepared enzyme-labeled antibody was used for preparing acalibration curve for human CRP by the same manner as in Example 1 (FIG.13). Ratio (S/N ratio) of the absorption of the specific coloration (10ng/ml of human CRP) to that of the blank (0 ng/ml of human CRP) was 1.25for the alkaline phosphatase of the present invention while that forCIAP was 5.13. Thus, the alkaline phosphatase of the present inventionhad less non-specific adsorption.

EXAMPLE 5

(1) Preparation of sheep anti-human CRP Fab' labeled with alkalinephosphatase.

A 0.1M phosphate buffer (pH: 6.0) (100 ml) containing 0.1M2-mercaptoethylamine and 10 mM EDTA was added to 1 ml of 0.1M phosphatebuffer (pH: 6.0) containing 5 mg of sheep anti-human CRP F(ab')₂(manufactured by Binding Site) and an incubation was conducted at 37° C.for 90 minutes. Said mixture was desalted with a 0.1M phosphate buffer(pH: 6.0) containing 5 mM EDTA and concentrated to 0.5 ml. On the otherhand, 10 μl of dimethylformamide containing 0.1 mg ofN-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate was addedto 500 μl of a 30 mM triethanolamine buffer (pH: 7.6; containing 1 mMMgCl₂ and 0.1 mM CoCl₂) containing 2.5 mg of the alkaline phosphatase ofthe present invention and an incubation was conducted at 30° C. for 30minutes. The resulting solution was desalted with a 0.1M Trishydrochloride buffer (pH: 7.0) containing 1 mM MgCl₂ and 0.1 mM CoCl₂and concentrated to 0.5 ml.

Maleimidated alkaline phosphatase was added to the sheep anti-human CRPFab' prepared as such and an incubation was conducted at 4° C. for 20hours. Then 50 μl of 10 mM 2-mercaptoethylamine was added thereto and anincubation was conducted at 25° C. for 20 minutes. The resulting mixturewas purified by Superdex™ 200 and the first peak was collected as anenzyme-labeled antibody.

(2) Calibration curve of human CRP.

Human CRP (0-1,000 ng/ml) (1 ml) was added to one polystyrene beadcoated with goat anti-human CRPIgG fractions (manufactured by NipponBiotest Laboratory) and an incubation was conducted at 30° C. for onehour. Then the solid phase was washed with PBS thrice, 1 ml of anenzyme-labeled antibody diluted to an extent of 1 U/ml in terms of thealkaline phosphatase activity and an incubation was conducted at 30° C.for one hour. This was further washed with PBS thrice, a 1Mdiethanolamine buffer (pH: 9.8) containing 11 mM p-nitrophenyl phosphateand 5 mM MgCl₂ was added, a reaction was conducted at 37° C. for 30minutes, then the reaction was stopped by adding 2 ml of 0.5N NaOHthereto and the absorbance at 405 nm was measured whereby a calibrationcurve was prepared (FIG. 14).

Comparative Example 3.

The same operations as in Example 2 were conducted for 500 μl of a 30 mMtriethanolamine buffer (pH: 7.6) (containing 1 mM MgCl₂, 0.1 mM ZnCl₂and 3M NaCl) containing 2.5 mg of CIAP to give an enzyme-labeledantibody.

The above-prepared enzyme-labeled antibody was used and a calibrationcurve for human CRP was prepared by the same manner as in Example 1(FIG. 14). Ratio (S/N ratio) of the absorbance of a specific coloration(10 ng/ml human CRP) to that of a blank (0 ng/ml human RRP) was 55.2 forthe alkaline phosphatase of the present invention while said ratio was34.8 for CIAP. Thus, the alkaline phosphatase of the present inventionshowed less non-specific adsorption.

EXAMPLE 6

(1) Preparation of an alkaline phosphatase labeled with streptoavidin.

Dimethylformamide (10 μl) containing 0.1 mg of S-acetylmercaptosuccinicanhydride was added to 600 μl of 0.1M phosphate buffer (pH: 7.5)containing 4 mg of streptoavidin and an incubation was conducted at 30°C. for 30 minutes. Then 20 μl of 1M EDTA (pH: 7.0), 120 μl of 0.1MTris/HCl (pH: 7.0) and 120 μl of 1M hydroxylamine hydrochloride wereadded thereto, an incubation was conducted at 30° C. for five minutes,the mixture was desalted with a 0.1M phosphate buffer (pH: 6.0) andconcentrated to 600 μl. The mercaptosuccinylated streptoavidin (100 μl)prepared as such was added to a solution of a maleimidated alkalinephosphatase prepared in Example 2, the mixture was incubated at 4° C.for 20 hours, the resulting mixture was purified by Superdex™ 200 andthe first peak was taken as an enzyme-labeled streptoavidin.

Comparative Example 4.

Enzyme-labeled streptoavidin was prepared from CIAP by the same methodas in Example 6.

Biotinyl BSA (0-5 ng) was applied to immobilon (Millipore), blocked withPBS containing 1% of casein and incubated with 0.3 U/ml of theenzyme-labeled streptoavidin of the present invention and CIAP-labeledstreptoavidin at 30° C. for one hour. Then this was made to react with a1M diethanolamine buffer (pH: 9.8; containing 5 mM MgCl₂) containing PPD(a luminous substrate) and a detection was conducted by sensitizing onan X-ray film. Both enzyme-labeled one of the present invention andCIAP-labeled one were able to detect 0.5 ng of biotinylatedstreptoavidin.

Then the enzyme-labeled streptoavidin of the present invention and theCIAP-labeled streptoavidin were stored at 40° C. for seven days in 50 mMTris/HCl (pH: 7.5) containing 1 mM MgCl₂ and the activities of alkalinephosphatase were compared. The results are given in FIG. 15 and theenzyme-labeled streptoavidin of the present invention was more stable(FIG. 15).

EXAMPLE 7

(1) Preparation of a biotinylated alkaline phosphatase.

Dimethyl formamide (20 μl) containing 0.128 mg ofD-biotinin-ε-aminocapric acid N-hydroxysuccinimide ester was added to600 μl of a 30 mM triethanolamine buffer (pH: 7.5) (containing 1 mMMgCl₂ and 0.1 mM CoCl₂) containing 30 mM of the alkaline phosphatase ofthe present invention and the mixture was stirred at 25° C. for threehours followed by dialyzing to a 50 mM Tris hydrochloride buffercontaining 0.1M NaCl, 1 mM MgCl₂, 0.1 mM CoCl₂ and 0.1% NaN₃.

Comparative Example 5.

Biotinylated alkaline phosphatase was prepared from CIAP by the samemethod as in Example 7.

Biotinyl BSA (0-5 ng) was applied by conventional means to immobilon(Millipore) and the mixture was blocked by PBS containing 1% of caseinand incubated at 30° C. for one hour with 1 μg/ml of the biotiylatedalkaline phosphatase of the present invention and 0.3 U/ml ofbiotinylated CIAP. After that, it was made to react with a 1Mdiethanolamine buffer (pH: 9.8) (containing 5 mM MgCl₂) containing aluminous substance (PPD) and a detection was conducted by sensitizingwith an X-ray film. Both the enzyme-labeled substance of the presentinvention and the CIAP-labeled one were able to detect 50 pg ofbiotinylated streptoavidin.

Then the thermal stabilities of alkaline phosphatase activity in 50 mMTris/HCl (pH: 7.5) containing 1 mM MgCl₂ were compared for thebiotinylated alkaline phosphatase of the present invention and for thebiotinylated CIAP. The results are given in FIG. 16 and the biotinylatedalkaline phosphatase of the present invention was stabler.

EXAMPLE 8

(1) Probe labeled with an alkaline phosphatase.

An oligonucleotide in which Uni-Link™ Amio Modifier (manufactured byClone Tech) was incorporated at 5'-terminal was synthesized by a commonmethod followed by purifying.

A disuccinimidylsuberic acid solution (10 mg/ml-DMSO) (50 μl) was addedto 10 μl of 0.1M NaHCO₃ containing 10 nmoles of the above probe, themixture was stirred, made to react at 25° C. for 15 minutes, subjectedto a gel filtration using a column of Sephadex G and the first peakcontaining oligonucleotide was collected. Said peak was concentrated to100 μl, then 40 μl of 0.1M NaHCO₃ containing 1.5 mg of the alkalinephosphatase of the present invention was added thereto and the mixturewas made to react at 25° C. overnight.

To said mixture was added about 500 ml of 20 mM Tris/HCl (pH: 7.0)containnig 1.0 mM MgCl₂ and a purification was conducted by means of ahigh performance liquid chromatography (eluting solution A: 20 mMTris/HCl of pH 7.0 containing 1.0 mM MgCl₂ ; eluting solution B: 20 mMTris/HCl of pH 7.0 containing 1.0 mM MgCl₂ and 1M NaCl) using MonoQ(made by Farmacia).

Comparative Example 6.

CIAP was treated by a method mentioned in Example 8 to prepare aCIAP-labeled probe.

The enzyme-labeled probe of the present invention and the CIAP-labeledprobe were treated with PBS containing 1 mM MgCl₂ at 70° C. for twohours and their alkaline phosphatase activities were compared. Theresults were, as shown in FIG. 17, that the probe labeled with theenzyme of the present invention was more stable.

What we claim is:
 1. An alkaline phosphatase having the followingphysical and chemical properties:(1) the alkaline phosphatase catalyzesthe following reaction:orthophosphoric acid monoester+H₂O→alcohol+orthophosphoric acid; (2) activators selected from the groupconsisting of Mg⁺⁺ and Co⁺⁺ ; (3) stabilizers selected from the groupconsisting of Mg⁺⁺ and Co⁺⁺ ; (4) a thermal stability wherein thealkaline phosphatase is stable at least for 30 minutes when treated atpH 7.5 and 60° C.; (5) a specific activity of at least 2,300 U/mg; (6)the alkaline phosphatase has no sugar chain; and (7) a gel filtrationmolecular weight from about 140,000 to about 150,000 and a SDS-PAGEmolecular weight from about 65,000 to about 67,000.
 2. An alkalinephosphatase having the following physical and chemical properties:(1)the alkaline phosphatase catalyzes the followingreaction:orthophosphoric acid monoester+H₂ O→alcohol+orthophosphoricacid; (2) activators selected from the group consisting of Mg⁺⁺ and Co⁺⁺; (3) stabilizers selected from the group consisting of Mg⁺⁺ and Co⁺⁺ ;(4) a thermal stability not higher than 60° C. at pH 7.5 for 30 minutes;(5) an optimum temperature not lower than 60° C.; (6) a stable pHwherein the pH is from about pH 6 to about 9 at 25° C. for 16 hours; (7)an optimum pH wherein the pH is from about pH 9 to about 10; (8) aspecific activity of at least 2,300 U/mg; (9) no sugar chain in thealkaline phosphatase; (10) a Km value of 0.34 mM (to p-nitrophenylphosphoric acid); (11) a gel filtration molecular weight from about140,000 to about 150,000 and a SDS-PAGE molecular weight from about65,000 to about 67,000; and (12) a substrate specificity wherein thealkaline phosphatase acts on substrates selected from the groupconsisting of p-nitrophenyl phosphate, 4-methyllumbelliferone phosphate,NADP, DL-α glycerophosphate, β-glycerophosphate, phenylphosphate,phosphoethanolamine and glucose-6-phosphate.
 3. An alkaline phosphatasehaving the following physical and chemical properties:(1) the alkalinephosphatase catalyzes the following reaction:orthophosphoric acidmonoester+H₂ O→alcohol+orthophosphoric acid; (2) activators selectedfrom the group consisting of Mg⁺⁺ and Co.sup. 30 +; (3) stabilizersselected-from the group consisting of Mg⁺⁺ and Co⁺⁺ ; (4) a thermalstability not higher than 60° C. at pH 7.5 for 30 minutes; (5) anoptimum temperature not lower than 60° C.; (6) a stable pH wherein thepH is between pH 6-9 at 25° C. for 16 hours; (7) an optimum pH whereinthe pH is between pH 9-10; (8) a specific activity of at least 2,300U/mg; (9) no sugar chain in the alkaline phosphatase; (10) a Km value of0.26 mM (to p-nitrophenyl phosphoric acid); (11) a gel filtrationmolecular weight from about 140,000 to about 150,000 and a SDS-PAGEmolecular weight from about 65,000 to about 67,000; and (12) a substratespecificity wherein the alkaline phosphatase acts on a substrateselected from the group consisting of p-nitrophenyl phosphate,4-methyllumbelliferone phosphate, NADP, DL-α glycerophosphate,β-glycerophosphate, phenylphosphate, phosphoethanolamine andglucose-6-phosphate.
 4. An alkaline phosphatase having the followingphysical and chemical properties:(1) the alkaline phosphatase catalyzesthe following reaction:orthophosphoric acid monoester+H₂O→alcohol+orthophosphoric acid; (2) activators selected from the groupconsisting of Mg⁺⁺ and Co⁺⁺ ; (3) stabilizers selected from the groupconsisting of Mg⁺⁺ and Co⁺⁺ ; (4) a thermal stability not higher than70° C. at pH 7.5 for 30 minutes; (5) an optimum temperature not lowerthan 60° C.; (6) a stable pH wherein the pH is from about pH 6 to about11; (7) an optimum pH wherein the pH is from about pH 9.5 to about 10;(8) a specific activity of at least 2,300 U/mg; (9) no sugar chain inthe alkaline phosphatase; (10) a Km value of 0.28 mM (to p-nitrophenylphosphoric acid); (11) a gel filtration molecular weight from about140,000 to about 150,000 and SDS-PAGE molecular weight from about 65,000to about 67,000; and (12) a substrate specificity wherein the alkalinephosphatase acts on a substrate selected from the group consisting ofp-nitrophenyl phosphate, 4-methyllumbelliferone phosphate, NADP, DL-αglycerophosphate, β-glycerophosphate, phenylphosphate,phosphoethanolamine glucose-1-phosphate and glucose-6-phosphate.